1. Field of the Invention
The present invention refers to a method for detection of the presence of molecules of interest in biological sources.
2. Description of the Related Art
Providing a method for detecting molecules of interest in biological sources is a need common to many areas of scientific research. In fact, the ability to identify the presence in biological samples of molecules, associated for example with clinical pathologies, has always represented an important objective for research, in particular in the field of medicine.
The bio-medical field is without doubt the main (although not the only) field in which this method is applied. In fact the inventions relating to methods with this purpose have been proposed with the aim of reaching an increasingly high level of sensitivity in detection, in particular at a diagnostic level.
The bio-chemical instrument most frequently used to detect the presence of specific molecules in cell or tissue extracts are antibodies, because of their extreme sensitivity and high binding specificity.
When an interaction with the molecule of interest occurs in these methods, it is highlighted using markers linked to the antibodies themselves, for example fluorescent substances, proteins with enzymatic activity, or radioactive markers.
The detection capacity of these markers has in any case been found to be considerably enhanced by the use of so-called signal amplification methods. These make it possible to obtain a stronger detection signal, for example by joining the marker not to the antibody used for specific recognition (primary antibody), but to a second antibody capable of binding the first one (secondary antibody). A number of methods have been developed using secondary antibodies, to which marker molecules are covalently bonded, for example fluorescent substances such as fluorescein or rhodamin, proteins with enzymatic activity such as alkaline phosphatase or horseradish peroxidase, in case of detection using electron microscope ferritin or colloidal gold spheres are also used.
Alternative amplification systems make use of the high binding affinity of molecules such as Biotin (a small, water-soluble vitamin) and streptavidin (bacterial protein), or those between lecithins (proteins capable of recognising and binding specific saccharide residues) and molecules such as glycoproteins, glycolipids or proteoglycans.
An extremely wide series of variations on this theme is possible, including those allowing creation of a network of markers, capable of considerably amplifying the detection signal. In any case all these methods are connected to the use of antibodies as molecules capable of specific identification of a target molecule, and they also have a sensitivity level that is limited by the concentration of target molecules.
The latter limitation has been overcome by methods drawn up more recently, which are based on the application of PCR (PolymeraseChain Reaction) techniques for this purpose, used here in place of the detection enzymes. These methods, in fact, are again based on the use of monoclonal antibodies against a specific ligand, which in this case, however, are not physically coupled to proteins, but to polynucleotides with a known sequence.
This makes it possible to perform subsequent detection by amplification of the known DNA sequence, which is effectively achieved using a polymerase chain reaction (PCR) (V. Ruzicka et al, 1993; T. Sano et al, 1992; H. Zhou et al, 1993).
This method, known as immuno PCR, makes it virtually possible to detect even a single antibody bound to the target molecule. In spite of the considerable improvement in sensitivity, these methods present a series of problems, among which the direct association of polynucleotides and antibodies, and the resulting high background due to the inevitable interference by the polynucleotide during binding of the antibody to the molecule of interest.
Furthermore, given that the specificity and yield of the amplification reaction depend on the temperature at which the reagents are mixed, it is possible to obtain, if assembly of the PCR reaction takes place at temperatures lower than those considered optimum for hybridisation of the primers, non-specific hybridisation products.
Currently this problem has been solved by addition to the amplification reaction of an essential reagent, only after the system has reached the temperature allowing specific hybridisation of the "Hot Start" primers. This procedure is achieved by mechanically separating a reagent (D. E. Birch et al, 1996), or by using antibodies to block the enzymatic activity of DNA polymerase (J. Cheng et al, 1996).
Recently, a PCR method performed on cells has also been developed, known as in situ PCR (G. J. Nuovo, 1994), in which amplification follows an in situ hybridisation reaction. According to this method, the DNA template and the primers are kept physically separate until the moment of cell lysis, with the advantage of avoiding any non-specific hybridisation reactions (EP 524808 Hoffmann La Roche Inc.).
Furthermore, there are a series of patents all relating to new diagnostic methods that are supposed to overcome many of the problems typical of current methods, and consequently to improve amplification of the response signal. These have as their subject matter various methods, such as the use of PCR associated with the discovery of particular proteins (WO9421676); the use of genetically engineered hybrid enzymes conjugated to ligands, for example epitopes of the virus HIV-1 (WO942636); the use of viral epitopes generated from nucleic acids combined with proteins binding the antigen (WO9406934); the use of parts of viral cDNA, for example taken from HCV, to express a viral epitope connected to a control sequence and a monoclonal antibody directed against the epitope. In the latter case the cDNA region of interest allows cross-reference controls to be carried out between the antibody response and that resulting after hybridisation with the DNA (EP388232).
Another patent, on the other hand, has as its subject the construction of a library of antigenic determinants, obtained by digestion with DNAase-I, of the genome of a virus, for example HIV, which is followed by expression of these fragments obtained by means of a suitable vector, and subsequent selection of said expression products by the use of antibodies (EP373070).